title: Molecular properties and fibril ultrastructure of types II and XI collagens in cartilage of mice expressing exclusively the α1(IIA) collagen isoform.
abstract: Until now, no biological tools have been available to determine if a cross-linked collagen fibrillar network derived entirely from type IIA procollagen isoforms, can form in the extracellular matrix (ECM) of cartilage. Recently, homozygous knock-in transgenic mice (Col2a1(+ex2), ki/ki) were generated that exclusively express the IIA procollagen isoform during post-natal development while type IIB procollagen, normally present in the ECM of wild type mice, is absent. The difference between these Col2a1 isoforms is the inclusion (IIA) or exclusion (IIB) of exon 2 that is alternatively spliced in a developmentally regulated manner. Specifically, chondroprogenitor cells synthesize predominantly IIA mRNA isoforms while differentiated chondrocytes produce mainly IIB mRNA isoforms. Recent characterization of the Col2a1(+ex2) mice has surprisingly shown that disruption of alternative splicing does not affect overt cartilage formation. In the present study, biochemical analyses showed that type IIA collagen extracted from ki/ki mouse rib cartilage can form homopolymers that are stabilized predominantly by hydroxylysyl pyridinoline (HP) cross-links at levels that differed from wild type rib cartilage. The findings indicate that mature type II collagen derived exclusively from type IIA procollagen molecules can form hetero-fibrils with type XI collagen and contribute to cartilage structure and function. Heteropolymers with type XI collagen also formed. Electron microscopy revealed mainly thin type IIA collagen fibrils in ki/ki mouse rib cartilage. Immunoprecipitation and mass spectrometry of purified type XI collagen revealed a heterotrimeric molecular composition of α1(XI)α2(XI)α1(IIA) chains where the α1(IIA) chain is the IIA form of the α3(XI) chain. Since the N-propeptide of type XI collagen regulates type II collagen fibril diameter in cartilage, the retention of the exon 2-encoded IIA globular domain would structurally alter the N-propeptide of type XI collagen. This structural change may subsequently affect the regulatory function of type XI collagen resulting in the collagen fibril and cross-linking differences observed in this study.
authors.affiliation: Department of Orthopaedic Surgery, Washington University, St Louis MO, USA; Department of Cell Biology & Physiology, Washington University, St Louis MO, USA.
authors.forename: Audrey
authors.initials: A
authors.lastname: McAlinden
authors.affiliation: Department of Orthopaedic & Sports Medicine, University of Washington, Seattle WA, USA.
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Traeger
authors.affiliation: Institute for Physiological Chemistry and Pathobiochemistry, University of Münster, Germany.
authors.forename: Uwe
authors.initials: U
authors.lastname: Hansen
authors.affiliation: Department of Orthopaedic & Sports Medicine, University of Washington, Seattle WA, USA.
authors.forename: Mary Ann
authors.initials: MA
authors.lastname: Weis
authors.affiliation: Department of Orthopaedic Surgery, Washington University, St Louis MO, USA.
authors.forename: Soumya
authors.initials: S
authors.lastname: Ravindran
authors.affiliation: Department of Orthopaedic Surgery, Washington University, St Louis MO, USA.
authors.forename: Louisa
authors.initials: L
authors.lastname: Wirthlin
authors.affiliation: Department of Orthopaedic & Sports Medicine, University of Washington, Seattle WA, USA.
authors.forename: David R
authors.initials: DR
authors.lastname: Eyre
authors.affiliation: Department of Orthopaedic & Sports Medicine, University of Washington, Seattle WA, USA. Electronic address: rjf@u.washington.edu.
authors.forename: Russell J
authors.initials: RJ
authors.lastname: Fernandes
mesh_terms: D000818:Animals; D002356:Cartilage / Q000378:metabolism / Q000648:ultrastructure; D020219:Chondrogenesis / Q000235:genetics*; D024043:Collagen Type II / Q000096:biosynthesis* / Q000235:genetics; D024161:Collagen Type XI / Q000235:genetics / Q000378:metabolism; D005109:Extracellular Matrix / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D051379:Mice; D008822:Mice, Transgenic; D020894:Microfibrils / Q000235:genetics / Q000648:ultrastructure; D059368:RNA Isoforms / Q000096:biosynthesis*
chemical_list: C447413:Col2a1 protein, mouse; D024043:Collagen Type II; D024161:Collagen Type XI; D059368:RNA Isoforms
keywords: Alternative splicing; Cartilage; Collagen cross-links; Collagen fibrils; Extracellular matrix; Heteropolymer; Post-translational modifications; Type IIA procollagen